Cover Page
The handle http://hdl.handle.net/1887/62776 holds various files of this Leiden University dissertation
Author: Peterse, Elisabeth
Title: Targeting chondrosarcoma and osteosarcoma cell metabolism : the IGF pathway and beyond
Date: 2018-06-13
Elisabeth F.P. Peterse
Targeting Chondrosarcoma and Osteosarcoma Cell Metabolism Elisabeth F.P. Peterse
Targeting Chondrosarcoma and Osteosarcoma
Cell Metabolism
The IGF pathway and beyond
UITNODIGING
voor het bijwonen van de openbare verdediging van
het proefschrift
Targeting Chondrosarcoma and Osteosarcoma
Cell Metabolism
door
Elisabeth F.P. Peterse
Woensdag 13 juni 16.15 Academiegebouw
Rapenburg 73 2311 GJ Leiden
Aansluitend bent u van harte welkom bij
De receptie om 17.15 de Waag
Aalmarkt 21 2311 EC Leiden De borrel om 20.30
Scheltema Marktsteeg 1 2312 CS Leiden
Paranimfen Emma brouwer en
Nienke de Kok
verdedigingelleke@gmail.com RSVP
Targeting Chondrosarcoma and Osteosarcoma Cell Metabolism
The IGF pathway and beyond
Elisabeth Francisca Patricia Peterse
ISBN: 978-94-6332-340-6
© Elisabeth F.P. Peterse, 2018. All rights reserved.
Layout and printing: GVO drukkers & vormgevers B.V.
Publication of this thesis was financially supported by the Department of Pathology, Leiden University Medical Center.
Targeting Chondrosarcoma and Osteosarcoma Cell Metabolism
The IGF pathway and beyond
Proefschrift
ter verkrijging van de graad van Doctor aan de Universiteit Leiden, op gezag van Rector Magnificus prof.mr. C.J.J.M. Stolker,
volgens besluit van het College voor Promoties te verdedigen op woensdag 13 juni 2018
klokke 16.15 uur
door
Elisabeth Francisca Patricia Peterse
geboren te Diessen
in 1991
Promotor: Prof. Dr. J.V.M.G. Bovée Co-promotor: Dr. A.M. Cleton-Jansen leden promotiecommissie: Prof. Dr. P.D.S. Dijkstra Dr. J.W. Wilmink
(Academic Medical Center, Amsterdam) Prof. Dr. A.B. Hassan
(University of Oxford, Oxford, United Kingdom) Prof. Dr. P.C.W. Hogendoorn
Table of contents
Chapter 1.
General introduction and outline of this thesis Chapter 2.
In vitro studies of osteosarcoma: A researcher’s perspective of quantity and quality
Chapter 3.
IR/IGF1R signaling as potential target for treatment of high-grade osteosarcoma
Chapter 4.
CORR Insights®: Transcriptional profiling identifies the signaling axes of IGF and transforming growth factor-β as involved in the pathogenesis of osteosarcoma
Chapter 5.
No preclinical rationale for IGF1R directed therapy in chondrosarcoma of bone
Chapter 6.
NAD synthesis pathway interference is a viable therapeutic strategy for chondrosarcoma
Chapter 7.
Targeting glutaminolysis in chondrosarcoma in context of the IDH1/2 mutation
Chapter 8.
Summary and future perspectives Chapter 9.
Nederlandse samenvatting Curriculum Vitae
List of publications Acknowledgements
9
35
43
63
69
91
113
137
153160 161163
Chapter 1
General introduction and outline
of this thesis
10 Chapter 1
Primary bone cancers are a heterogeneous group of cancers that originate from the bone. They account for less than 0.2 % of all cancers (1). In the Netherlands, 228 primary bone cancers have been diagnosed in 2016 (2).
This heterogeneous group of cancers differ in terms of etiology, clinical manifestation and prognosis. Here, we focus on the two most common subtypes: osteosarcoma and chondrosarcoma.
Osteosarcoma
Osteosarcoma is the most frequent primary malignant bone tumour. This tumour is characterized by the production of osteoid matrix. The incidence of osteosarcoma has a bimodal distribution, with the first peak around puberty and the second, smaller peak, observed above the age of 60. The incidence is 5.0 (4.6-5.6) per million 0-19 year olds (3, 4). The second peak in incidence is likely to represent a secondary malignancy, as half of the osteosarcoma patients above 60 years suffer from Paget disease of the bone (5). In addition, osteosarcoma is the most common radiation-induced sarcoma (5-7). Most primary osteosarcomas occur in the metaphysis of the long bones, in particular the distal femur (30%), the proximal tibia (15%) and the proximal humerus (15%), where bone growth is particularly active, especially at the pubertal age of onset of this tumour (4, 5).
Treatment of osteosarcoma consists of pre-operative and postoperative chemotherapy in combination with surgical resection of the tumour. Limb salvage has largely replaced amputation for local tumour control. Several chemotherapeutic agents have been tested in clinical trials, in which the currently used combination of doxorubicin, cisplatin and methotrexate was found to be the most effective (8). Despite the harsh chemotherapeutic regime, about 45% of the patients develop distant metastases, most often in the lungs (85%) (8, 9). Since the introduction of the chemotherapy in the 1980s survival has reached a plateau of 65-70% (10, 11). Therefore, new treatment options for osteosarcoma are desperately needed.
Osteosarcoma can be divided in several subtypes (5, 12). The focus of this thesis is on conventional osteosarcoma. Conventional osteosarcoma can be further
11
1
General introduction and outline of this thesis
subdivided, based on the predominant matrix, in the osteoblastic (76-80% of the cases), the chondroblastic (10-13%) and the fibroblastic (10%) variant (5, 13, 14). Less common subtypes of conventional osteosarcoma are the giant cell rich, osteoblastoma-like, epithelioid, clear cell and chondroblastoma- like subtype (5). Although the proportion of good histological responders, defined as >90% chemotherapy induced tumour necrosis, differs significantly between the conventional osteosarcoma subtypes, there is no difference in the overall survival (14).
Osteosarcoma cell of origin
The cell of origin of osteosarcoma is not well defined (15). However, two candidate progenitor cells are discussed in literature. The first hypothesis is that osteosarcoma arises from mesenchymal stem cells (MSCs). MSCs have the capability to differentiate into multiple lineages, such as myocytes, adipocytes, chondrocytes and osteocytes. These lineages of differentiation are respectively initiated by MyoD, PPARγ, SOX9, and osterix/Runx2 (16, 17). The capacity of MSCs to differentiate in multiple lineages resembles the histological spectrum observed in osteosarcoma (18). In addition, it has been suggested that the timing of the mutation can explain the different stages of differentiation observed in osteosarcoma, where an early genetic or epigenetic alteration may result in the development of an highly aggressive, undifferentiated osteosarcoma and vice versa (17, 19). Further evidence favouring the theory that osteosarcoma arises from MSCs comes from in vitro and in vivo studies, and from observations in the clinic. In vitro, our group and others have shown that mouse MSCs can undergo spontaneous transformation after long time culturing in vitro (18, 20- 23) and that these MSCs formed osteosarcomas upon in vivo grafting (18).
However, human MSCs do not show spontaneous in vitro transformation, even after prolonged culturing (24-27). In addition, publications reporting spontaneous human stem cell transformation have been retracted (28, 29) and can most likely be attributed to cross-contamination. Clinical observations also provide evidence favouring the mesenchymal stem cell theory, as osteosarcoma formation is found in patients following bone-marrow transplantation for unrelated diseases (30, 31). In addition, the existence of primary extraskeletal osteosarcoma supports this theory as in this rare subtype, osteosarcoma arises in tissues where no osteoblasts are present. The second theory hypothesizes
12 Chapter 1
that osteosarcomas arise from osteoblast-committed cells. Evidence for this theory stems mostly from studies in mice that show that osteoblast specific knockout of p53 (and Rb) can induce murine osteosarcoma (32-36). More specifically, Walkley et al. and Berman et al. made an osterix-cre-mediated deletion of p53 and pRb, which resulted in osteosarcoma formation (32, 35). As mentioned above, osterix is a transcription factor that is required for osteoblast differentiation and bone formation (37). Interestingly, p53 (and Rb) knockdown in undifferentiated mesenchymal cells also results in the formation of several sarcomas, among which osteosarcoma is the most frequent one (33). One study by Quist et al. compared osteosarcoma formation in Prx1-Cre, Collagen-1α1-Cre and Osteocalcin-Cre conditional disruption of p53 and Rb to transform undifferentiated mesenchyme, preosteoblasts and mature osteoblast, respectively (36). The Prx1 and Collagen-1α1 had a near complete penetrance of osteosarcoma, whereas only 44% of the osteocalcin-cre mice mice developed osteosarcomas (36). Although there seems to be increasing evidence supporting the first hypothesis, the differentiation state of the progenitor cells remain a point of debate among those who study this disease.
The IGF pathway
The IGF pathway has two closely related ligands: IGF1 and IGF2. IGF2 is an imprinted gene, only expressed from the paternal allele; IGF1 is mainly produced by the liver upon stimulation by the growth hormone (GH). The GH is produced by the pituitary gland and under control of the hypothalamus, as the hypothalamus produces the growth hormone releasing hormone (GHRH) and the growth hormone-inhibiting hormone (GHIH) (also known as somatostatin) which balance determines the amount of GH that is released. Besides the function of IGF1 and IGF2 as endocrine hormones, they can also act in autocrine and paracrine manners (38). In the circulation, six IGF binding proteins (IGFBPs) can bind IGF1 and IGF2 (39). IGFBPs are expressed in many peripheral tissues, except for IGFBP1 which is mainly produced by the liver (40). These binding proteins increase the half-lives of the IGF ligands, but they also interfere in the interaction between the IGF ligands and their receptors. IGFBP3 is the most abundant protein, which forms a ternary complex with insulin-like growth factor acid-labile subunit (IGFALS) and accounts for 80% of all IGF binding.
IGF1 and IGF2 can bind to the IGF1 receptor (IGF1R). Unlike IGF1, IGF2
13
1
General introduction and outline of this thesis
can additionally bind to the insulin receptor (IR) with high affinity (Figure 1). Two monomeric isomers of the IR have been identified: IRA (lacking exon 11) and IRB (with exon 11). These alternative splice variants differ in the C-terminus, where they influence receptor-ligand interaction (41). IGF2, in addition to binding to the IR and the IGF1R, can bind the IGF2 receptor (IGF2R). The IGF2R does not confer intracellular signalling but functions as a decoy receptor to decrease the availability of IGF2 to the IGF1R (42). Upon ligand binding, the IGF1R can form homodimers or hybrid receptors with the IR. The α chains of the IGF1R (which are located extracellular) induce tyrosine autophosphorylation of the β chains (which span the membrane) (43). This phosphorylation recruits the downstream signalling protein insulin receptor substrate (IRS) to the cell membrane (43). Phosphorylation of IRS creates binding sites for SH2 domain of various proteins, which finally leads to the activation of the PI3K/AKT/mTOR pathway and the Ras/Raf/ERK signalling pathways (43). Both these pathways are involved in many cellular functions, such as cell growth, proliferation and differentiation (44).
Indications for IGF pathway involvement in osteosarcoma genesis
In normal development, IGF1R signalling plays a key role in the growth and development of bone. This is illustrated in mice, as IGF1R knockout mice are approximately 55% smaller compared to IGF1R wildtype mice (45).
Interestingly, in dogs, the size of different breeds is dependent on IGF1 plasma levels (46). Due to the role of the IGF pathway in normal bone development, the IGF pathway has been studied for decades in osteosarcoma. Interestingly, the peak incidence of osteosarcoma correlates with the increased levels of GH and IGF ligands in puberty (Figure 2). Furthermore, two studies suggest that height at diagnosis is a risk factor of osteosarcoma (47, 48). In addition, a few cases of osteosarcoma in patients with acromegaly are reported (49) and patients with a congenital IGF1 deficiency seem protected against cancer (50). Multiple GH treated patients presented with osteosarcoma (51, 52).
Strikingly, a recent study identified mutations in IGF signalling genes in 7%
and IGF1R amplification in 14% of osteosarcoma cases (53). These clinical observations combined with the role of the IGF pathway in normal bone development strongly suggest a role for the IGF pathway in osteosarcoma genesis.
14 Chapter 1
Figure 1. The components of the IGF pathway, adapted from van Maldegem et al. (54).
15
1
General introduction and outline of this thesis
Chondrosarcoma
Chondrosarcoma is the second most common type of malignant bone tumour.
It accounts for 20% of all bone cancers and is characterized by the production of cartilage. It is most common in the 3rd to 6th decade of life (Figure 2), and most frequently occurs in the bones of the pelvis, the ribs, and bones of the extremities. In rare cases, they occur in the small bones of the hands and feet, or in the skull (56). Conventional chondrosarcoma is the most common subtype that accounts for 85% of the cases (57). This subtype can be histologically subdivided into atypical cartilaginous tumour (ACT), grade II and grade III chondrosarcomas (58). The ACT, previously known as grade I, accounts for 61%
of the cases. The first line treatment is curettage with local adjuvant treatment, resulting in a 5-year survival rate of 95%. Grade II (36%) and grade III (3%) chondrosarcomas have a worse 5-year survival of 86% and 58%, respectively, due to the occurrence of metastases (59-61). These tumours are treated by en bloc resection with wide resection margins to prevent local recurrence.
Chondrosarcoma patients with unresectable disease, due to tumour location, tumour size or extensive metastatic disease, have a 5 year survival of only 2%
(62, 63). These chondrosarcoma patients slightly benefit from doxorubicin- based chemotherapy, which increases the 3 year survival from 8% to 26% (62).
Chondrosarcoma either arises in the metaphysis or epiphysis of the bones, in which case it is called a central chondrosarcoma, or it arises on the surface
0 20 40 60 80
0 2 4 6 8 10
0 200 400 600
Age
Incidence rates per 1 million Median IGF1 Males (ng/ml)
Chondrosarcoma incidence IGF1 Levels
Osteosarcoma incidence
Figure 2. The peak in IGF1 levels correlates to the peak incidence in osteosarcoma.
Osteosarcoma incidence rates, chondrosarcoma incidence rates and IGF1 levels were derived from Mirabello et al. (55), WHO Classification of bone tumours 2013 and the Mayo Clinic, respectively.
16 Chapter 1
of the bone, in which case it is called a peripheral chondrosarcoma. Central and peripheral differ in aetiology, demonstrated by their different genetic background (Figure 3).
Central chondrosarcomas
In addition to primary chondrosarcoma, where tumours arise without a precursor lesion, secondary chondrosarcomas can also arise, in which the tumour arises from a benign precursor lesion. In case of a central chondrosarcoma, this benign precursor lesion is the enchondroma (Figure 3). Enchondromas show a predilection for the small bones of the hands and feet, and occur over a wide age distribution (64). Ollier disease and Maffucci syndrome, where patients present with multiple enchondromas, suggest the presence of an underlying genetic alteration. Indeed, gain of function mutations in isocitrate dehydrogenase 1 and -2 (IDH1 and -2) have been identified as driver mutations of enchondromas (65-67). 52%- 87% of the enchondromas harbour an IDH1/2 mutation (65, 66).
IDH mutation
p53, CDKN2A silencing, Rb
(Enchondroma)
Low-grade central chondrosarcoma
High-grade central chondrosarcoma EXT mutation
Osteochondroma
Low-grade peripheral chondrosarcoma
High-grade peripheral chondrosarcoma
Figure 3. Chondrosarcoma genetics. EXT mutations have been identified as potential driver mutations of osteochondromas, the benign precursor lesions of peripheral chondrosarcomas.
IDH1/2 mutations can give rise to enchondromas, which can than develop into central chondrosarcomas. However, central chondrosarcomas can also occur without the presence of a benign precursor lesion. Mutations in p53 and retinoblastoma (Rb) and CDKN2A silencing have been identified in the development of low-grade to high-grade lesions.
17
1
General introduction and outline of this thesis
IDH1 and IDH2 are essential enzymes in cell metabolism, as they convert isocitrate to α-ketoglutarate (α-KG) in respectively the cytoplasm and the mitochondria (Figure 4). The mutant enzyme acquires the activity to convert α-KG to D-2-hydroxyglutarate (D-2-HG), an oncometabolite that competitively inhibits the α-KG dependent enzymes by the high structural similarities (68). More specifically, D-2-HG inhibits histone demethylases and tet methylcytosine dioxygenase 2 (TET2)(68). TET2 is essential
Figure 4. Schematic representation of the cellular consequences of the IDH1 and the IDH2 mutation. Mutations in IDH1/2 result in the production of D-2-HG. D-2-HG inhibits histone demethylases and TET2. TET2 catalyses the conversion 5-methylcytosine to 5-hydroxymethylcytosine, the first step in DNA demethylation.
18 Chapter 1
for the conversion of the modified genomic base 5-methylcytosine to 5-hydroxymethylcytosine, the first step in cytosine demethylation. An aberrant methylation phenotype is observed in IDH1/2 mutated gliomas (69- 72), leukaemia’s (73) and enchondromas (66).
Peripheral chondrosarcomas
Peripheral chondrosarcomas always arise secondary from osteochondromas.
Osteochondromas are benign lesions, characterized by bony projections with a mature hyaline cartilaginous cap. They are most commonly located adjacent to the growth plate of the long bones (Figure 3) (74). Malignant transformation from a solitary osteochondroma to a secondary peripheral chondrosarcoma only occurs in 1% of the cases. Multiple osteochondromas are found in approximately 15% of the patients (75). When this is the case a patient has the hereditary autosomal dominant syndrome ‘Multiple Osteochondromas’
(MO), which is caused by a germline mutation in the exostosin-1 (EXT1) or – 2 (EXT2) gene (74-76). In the 10-15% of the multiple osteochondroma patients where no point mutation in EXT1 or EXT2 can be identified, somatic mosaicism with large genomic deletions is likely the underlying mechanism for osteochondroma genesis(77, 78). Somatic homozygous deletions of EXT1 can also be found in sporadic osteochondromas (74, 75). Strikingly, secondary peripheral chondrosarcomas no longer contain the EXT mutations, suggesting that these arise from the wildtype EXT cells present in the lesion (79).
EXT1 and EXT 2 are essential for heparan sulfate synthesis as they catalyse the elongation of the heparan sulfate chains. Heparan sulfate is a proteoglycan that regulates Indian Hedgehog (IHH) signalling in the growth plate (80, 81). IHH acts together with the parathyroid hormone-related protein in a negative feedback mechanism that regulates the balance between chondrocyte proliferation and differentiation in the growth plate (81-83). When IHH signalling is impaired due to a mutation in EXT1 or EXT2, this results in less chondrocyte differentiation and increased chondrocyte proliferation, thereby causing osteochondroma genesis.
Rare chondrosarcoma subtypes
Dedifferentiated chondrosarcoma accounts for 10% of chondrosarcoma
19
1
General introduction and outline of this thesis
patients (84 ). It is a highly malignant variant, reflected by the 28% 10-year survival that is seen in patients diagnosed without metastasis (85). Patients that are diagnosed with metastasis have an even worse prognosis, as only 10% survives the first two years (85). Dedifferentiated chondrosarcoma has a typical histology, as two clearly distinct compartments can be observed.
The first is a well-differentiated cartilage tumour, usually an enchondroma or a low-grade conventional chondrosarcoma. This component is juxtaposed to a dedifferentiated, high-grade non-cartilaginous sarcoma (86). The dedifferentiated component determines patient prognosis, and is therefore the focus of research that aims to identify new therapeutic strategies.
In addition to genetic alterations that are present in many cancer types, such as p53 mutation, PTEN deletion, MYC amplification and MDM2 amplification (86-88), mutations in IDH1/2 have been identified in 54% of the dedifferentiated chondrosarcomas (86).
Mesenchymal chondrosarcoma is a rare aggressive subtype in which distant metastasis can be identified even after 20 years (89-91). It accounts for 3% of all chondrosarcoma cases, and has a 10-year survival of 43% (92). Small round undifferentiated cells with islands of differentiated cartilage histologically characterize this subtype. In addition to its occurrence in the skeleton (mainly in the craniofacial bones, the ribs and the vertebrae), 20-30% of the cases present with primary soft tissue localization (64, 89). Tumour localization in the jaw is a bad prognostic factor, as well as presentation with metastases, while a young age is potentially a good prognostic factor (93, 94).
In contrast to dedifferentiated chondrosarcomas, no mutations in IDH1/2 have been identified. Furthermore, mutations in p53, deletions in p16 and MDM2 amplifications are rare events (86, 95, 96). Interestingly, mesenchymal chondrosarcomas carry the HEY1-NCOA2 fusion gene (97). Furthermore, in a case where this fusion was absent, an IRF2BP2-CDX1 fusion gene has been identified (98). This demonstrates that mesenchymal chondrosarcoma has a distinct genetic background.
Clear cell chondrosarcoma is a low-grade variant of chondrosarcoma, with a mortality of ~15% (99). It is the most rare subtype, as it accounts for only 2% of
20 Chapter 1
all chondrosarcoma cases. While metastases are rare, clear cell chondrosarcomas recur locally after curettage in 86% of the cases (100, 101). Interestingly it has a male: female ratio of 2.6:1, and is therefore the only subtype that has a sex preference (100). It derived its name by the histological presentation, showing clear cells that are surrounded by hyaline cartilage (99). It is most commonly located at the epiphyseal ends of long bones, and in line with conventional chondrosarcoma, the best treatment option is en bloc resection with clear margins (99, 100). Clear cell chondrosarcomas have widespread chromosome gains and losses, often with hemizygous involvement of the p16 locus. No alterations in IDH1/2, p53 and MDM2 have been reported (86).
Chondrosarcoma cell of origin
Originally, it was believed that chondrosarcoma originates from remnants of the growth plate cartilage. Although the chondrosarcoma cell of origin remains uncertain, it is now widely supported that chondrosarcomas most likely originate from MSCs undergoing chondrogenic differentiation (102).
The formation of chondroid callus during bone facture healing demonstrates the lifetime presence of these cells in bones. Furthermore, the existence of extraskeletal chondrogenic neoplasms suggests that these cells are even present outside the skeleton (103). A strong argument favouring the MSC as the cell of origin for central chondrosarcoma is provided by a study from our group, in which elevated levels of D-2-HG (the oncometabolite induced by an IDH1/2 mutation) blocked osteogenic differentiation and variably promoted chondrogenic differentiation of MSCs (104). For secondary peripheral chondrosarcoma, compelling evidence comes from studies in zebrafish and mice. Chondrocytes in Ext2-null zebrafish have been shown not to undergo terminal differentiation, and pre-osteoblasts failed to differentiate towards osteoblasts (105). In mice, Ext1 inactivation in chondrocytes resulted in osteochondromas formation, the precursor lesion of secondary peripheral chondrosarcomas, thereby demonstrating that osteochondromas originate from proliferating chondroctyes in the growth plate cartilage (106, 107).
There are striking parallels between normal cartilage growth and differentiation and cartilage tumours (108). Histologically, distinct chondrosarcoma subtypes show high similarities with different zones of chondrocyte differentiation in the
21
1
General introduction and outline of this thesis
normal growth plate (106). In addition, an analyses that evaluated expression levels of chondrogenesis-relevent genes demonstrated that grade I tumours cluster with chondrocytes, while grade III tumours cluster with earlier stages of MSCs (107). Due to these parallels, increasing our understanding of normal cartilage development will increase our understanding of chondrosarcoma genesis.
Targeting chondrosarcoma cell metabolism
Already in 1924, Otto Heinrich Warburg observed that cancer cells metabolize glucose in a different way than normal differentiated cells: while normal cells use mitochondrial oxidative phosphorylation to produce their energy, tumour cells mainly produce their energy via glucose fermentation, even in the presence of sufficient oxygen (110, 111). This phenomenon, later called “the Warburg effect”, was the first indication that cancer cell metabolism differs from normal cell metabolism. The aerobic glycolysis used by cancer cells is an inefficient way to generate adenosine 5’-triphosphate (ATP), but might be required to allow the production of nucleotides, amino acids and lipids, which are essential for cell proliferation (112). Despite the fact that the Warburg effect was identified almost a decade ago, relatively little progress had been made in increasing the understanding of how altered cell metabolism contributes to carcinogenesis. However, the recent identification of mutations in TCA cycle enzymes in several cancers, among which mutations in IDH1 and IDH2, renewed the interests in cancer cell metabolism as this demonstrates that enzymes involved in cell metabolism play an important role in tumourigenesis.
In 2011, Hanahan and Weinberg recognized the reprogramming of energy metabolism as a hallmark of cancer (113). The identification of mutations in TCA cycle enzymes opened new therapeutic possibilities.
When AGI-5198 was identified as a specific IDH1 mutant inhibitor (selective for the R132H mutation) (114), the field of chondrosarcoma immediately recognized its potential as chondrosarcoma therapy. However, our group has shown that treatment with AGI-5198 does not influence the tumorigenic properties of chondrosarcoma cells (115). In IDH2 mutant leukaemia, an initial proliferation burst followed by cellular differentiation was observed upon inhibition of the IDH2 mutant enzyme (116, 117). Furthermore, in IDH1 mutant glioma, the initially described impaired cell proliferation upon IDH1 mutant inhibition (114) could not be confirmed in other studies (118, 119).
22 Chapter 1
In contrast to gliomas (120-122), the IDH1/2 mutation status does not correlate with chondrosarcoma survival, and does not cause loss of 5-hydroxymethylcytosine or altered histone modifications in central chondrosarcomas (123). This suggests that while the IDH1 or -2 mutations are an early event in tumorigenesis, at later stages other processes involved in chondrosarcoma progression render these cells independent of the mutant IDH1/2 enzymes. Therefore, we propose to target the altered metabolism caused by the IDH1/2 mutations as therapeutic strategy for chondrosarcoma.
One pathway that is linked to cell metabolism and has been suggested to play a role in chondrosarcoma progression is the IGF pathway (124). IGFBP3, the binding protein that accounts for 80% of the IGF1 and IGF2 binding, is lower expressed in enchondromas than in normal growth plate cartilage, and expression levels decrease with increasing grades of chondrosarcoma (125). Furthermore, it has been described that IGF1 induces migration of chondrosarcoma cell lines, which can be blocked by an IGF1R antibody (126). Together with collaborators from the Ludwig Center at Dana-Faber/Harvard, our group performed functional profiling of Receptor Tyrosine Kinases in chondrosarcomas (127). IGF1Rβ and IRβ hypermethylation was identified in 1 out of 5 cell lines tested; an IGF1R/
IR inhibitor could inhibit proliferation of this cell line (127). Taken together, these studies suggest a potential role for the IGF pathway in chondrosarcoma development, migration and proliferation.
It has been described that the IDH1/2 mutation influences nicotinamide adenine dinucleotide (NAD+) synthesis. More specifically, metabolic profiling of glioblastoma cells upon mutant IDH1 inhibition revealed nicotinamide phosphoribosyltransferase (NAMPT) as a therapeutic target for IDH1/2 mutated tumours (128). An increased sensitivity for NAMPT inhibitors correlated with a decreased expression of nicotinic acid phosphoribosyltransferase (NAPRT) caused by an increased methylation of the NAPRT promoter (128). NAMPT and NAPRT are rate-limiting enzymes of the primary salvage pathway and the Preiss-handler pathway, respectively, which are involved in NAD+ synthesis. Tumour cells depend on these pathways for their rapid NAD turnover, as they lack expression of key enzymes in the de novo synthesis of NAD+ from tryptophan (129-131).
23
1
General introduction and outline of this thesis
Another main metabolic pathway that has been identified as being altered by the IDH1/2 mutations is glutaminolysis (132-137), an important energy- producing pathway in tumour cells. Glutamine, a non-essential amino acid, can subsequently be converted to glutamate and α-ketoglutarate by glutaminase and glutamate dehydrogenase, respectively, thereby fuelling the TCA cycle and the D-2-HG production in IDH1/2 mutated cells. IDH1/2 mutated cells are more sensitivity to several compounds that interfere with glutaminolysis compared to IDH1/2 wildtype cells, such as CB-839, metformin and phenformin. CB-839 is an inhibitor of glutaminase that decreases D-2-HG levels and inhibits growth of IDH1/2 mutated acute myeloid leukaemia cells (138). An endogenous heterozygous knock-in of the R132H IDH1 mutation sensitizes breast epithelial cells to metformin, the first in line antidiabetic drug, and its lipophilic analogue phenformin (133). Therefore, inhibition of NAD+ synthesis or glutaminolysis potentially leads to synthetic lethality with the mutated IDH1/2 enzyme in chondrosarcoma.
Aim and Outline of the thesis
The aim of this thesis was to develop new therapeutic strategies by identifying cellular pathways that are essential for chondrosarcoma and osteosarcoma cell survival.
Chapters 2, 3 and 4 are focussed on osteosarcoma. Although osteosarcoma is a rare disease, many preclinical experiments with osteosarcoma cells have been published. In Chapter 2, a systemic analysis of the literature on osteosarcoma in vitro studies is given to determine how the quantity of these studies developed over time. The downside of this huge number of studies is discussed from a researcher’s perspective. Chapter 3 reports on the analyses of genome-wide gene expression data, where overexpression of the IGF pathway in osteosarcoma is identified compared to osteoblasts and MSCs.
Osteosarcoma cell lines were treated with an IGF1R/IR dual inhibitor.1 In Chapter 4, a commentary on a similar transcriptional profiling study is given.
An overview of where we are with IGF1R-directed therapy, where we need to go and how we get there is presented.
1. The gene set analysis described in Chapter 3 was performed by dr. M.L. Kuijjer.
E.F.P. Peterse performed the Western blotting and the Proliferation assays.
24 Chapter 1
Chapters 5, 6 and 7 are focussed on chondrosarcoma. In Chapter 5, the potency of targeting the IGF pathway in chondrosarcoma is explored, as previous studies suggested the IGF pathway as a potential therapeutic target.
IGF1R expression levels are evaluated in cartilage tumours, and expression of IGF1R signalling mediators and sensitivity for IGF1R inhibition is evaluated in chondrosarcoma cell lines. In chapters 6 and 7, metabolic pathways altered by the IDH1/2 mutation are explored as therapeutic strategy. In Chapter 6, interference in NAD+ synthesis pathway is evaluated, as vulnerability for NAD+ depletion is reported for IDH1/2 mutant cells. Chondrosarcoma cell lines were treated with NAMPT inhibitors, and NAPRT expression and methylation was evaluated in cartilage tumours using qRT-PCR and genome-wide methylation arrays. Chapter 7 describes a preclinical study evaluating the effect of interfering in chondrosarcoma cell glutaminolysis.
The expression of glutaminase, the first essential enzyme of glutaminolysis, is compared between low-grade and high-grade chondrosarcomas to see if this pathway is involved in chondrosarcoma progression. In addition, chondrosarcoma cell lines are treated with the glutaminase inhibitor CB-839, and the compounds metformin, phenformin and chloroquine that all inhibit glutaminolysis at different levels. In Chapter 8, results described in chapters 2 to 7 are discussed and future perspectives are given.
25
1
General introduction and outline of this thesis
References
1. Giuffrida AY, Burgueno JE, Koniaris LG, Gutierrez JC, Duncan R, Scully SP.
Chondrosarcoma in the United States (1973 to 2003): an analysis of 2890 cases from the SEER database. J Bone Joint Surg Am 2009;91(5):1063-72.
2. The Netherlands Cancer Registry. In. http://www.cijfersoverkanker.nl.
3. Ottaviani G, Jaffe N. The epidemiology of osteosarcoma. Cancer Treat Res 2009;152:3-13.
4. Savage SA, Mirabello L. Using epidemiology and genomics to understand osteosarcoma etiology. Sarcoma 2011;2011:548151.
5. Rosenberg AE, Cleton-Jansen A-M, de Pinieux G, Deyrup AT, Hauben E, Squire J.
Conventional Osteosarcoma. World Health Organisation Classification of Tumours of Soft Tissue and Bone: IARC Press; Lyon, 2013, pp 282-288.
6. Wiklund TA, Blomqvist CP, Raty J, Elomaa I, Rissanen P, Miettinen M. Postirradiation sarcoma. Analysis of a nationwide cancer registry material. Cancer 1991;68(3):524-31.
7. Brady MS, Gaynor JJ, Brennan MF. Radiation-associated sarcoma of bone and soft tissue. Arch Surg 1992;127(12):1379-85.
8. Anninga JK, Gelderblom H, Fiocco M, Kroep JR, Taminiau AH, Hogendoorn PC, et al. Chemotherapeutic adjuvant treatment for osteosarcoma: where do we stand? Eur J Cancer 2011;47(16):2431-45.
9. Pakos EE, Nearchou AD, Grimer RJ, Koumoullis HD, Abudu A, Bramer JA, et al.
Prognostic factors and outcomes for osteosarcoma: an international collaboration. Eur J Cancer 2009;45(13):2367-75.
10. Smith MA, Seibel NL, Altekruse SF, Ries LA, Melbert DL, O’Leary M, et al. Outcomes for children and adolescents with cancer: challenges for the twenty-first century. J Clin Oncol 2010;28(15):2625-34.
11. Gatta G, Botta L, Rossi S, Aareleid T, Bielska-Lasota M, Clavel J, et al. Childhood cancer survival in Europe 1999-2007: results of EUROCARE-5--a population-based study. Lancet Oncol 2014;15(1):35-47.
12. Mohseny AB. Bone: Conventional osteosarcoma. Atlas of Genetics and Cytogenetics in Oncology and Haematology 2008.
13. Bacci G, Longhi A, Versari M, Mercuri M, Briccoli A, Picci P. Prognostic factors for osteosarcoma of the extremity treated with neoadjuvant chemotherapy: 15-year experience in 789 patients treated at a single institution. Cancer 2006;106(5):1154-61.
14. Hauben EI, Weeden S, Pringle J, Van Marck EA, Hogendoorn PC. Does the histological subtype of high-grade central osteosarcoma influence the response to treatment with chemotherapy and does it affect overall survival? A study on 570 patients of two consecutive trials of the European Osteosarcoma Intergroup. Eur J Cancer 2002;38(9):1218-25.
15. Mutsaers AJ, Walkley CR. Cells of origin in osteosarcoma: mesenchymal stem cells or osteoblast committed cells? Bone 2014;62:56-63.
16. Mohseny AB, Cai Y, Kuijjer M, Xiao W, van den Akker B, de Andrea CE, et al. The activities of Smad and Gli mediated signalling pathways in high-grade conventional osteosarcoma. Eur J Cancer 2012;48(18):3429-38.
17. Tang N, Song WX, Luo J, Haydon RC, He TC. Osteosarcoma development and stem cell differentiation. Clin Orthop Relat Res 2008;466(9):2114-30.
18. Mohseny AB, Szuhai K, Romeo S, Buddingh EP, Briaire-de Bruijn I, de Jong D, et al. Osteosarcoma originates from mesenchymal stem cells in consequence of aneuploidization and genomic loss of Cdkn2. J Pathol 2009;219(3):294-305.
26 Chapter 1
19. Mohseny AB, Hogendoorn PC. Concise review: mesenchymal tumors: when stem cells go mad. Stem Cells 2011;29(3):397-403.
20. Miura M, Miura Y, Padilla-Nash HM, Molinolo AA, Fu B, Patel V, et al. Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation. Stem Cells 2006;24(4):1095-103.
21. Tolar J, Nauta AJ, Osborn MJ, Panoskaltsis Mortari A, McElmurry RT, Bell S, et al.
Sarcoma derived from cultured mesenchymal stem cells. Stem Cells 2007;25(2):371-9.
22. Xu S, De Becker A, De Raeve H, Van Camp B, Vanderkerken K, Van Riet I. In vitro expanded bone marrow-derived murine (C57Bl/KaLwRij) mesenchymal stem cells can acquire CD34 expression and induce sarcoma formation in vivo. Biochem Biophys Res Commun 2012;424(3):391-7.
23. Zhou S, Li F, Xiao J, Xiong W, Fang Z, Chen W, et al. Isolation and identification of cancer stem cells from human osteosarcom by serum-free three-dimensional culture combined with anticancer drugs. J Huazhong Univ Sci Technolog Med Sci 2010;30(1):81-4.
24. Bernardo ME, Zaffaroni N, Novara F, Cometa AM, Avanzini MA, Moretta A, et al.
Human bone marrow derived mesenchymal stem cells do not undergo transformation after long-term in vitro culture and do not exhibit telomere maintenance mechanisms.
Cancer Res 2007;67(19):9142-9.
25. Choumerianou DM, Dimitriou H, Perdikogianni C, Martimianaki G, Riminucci M, Kalmanti M. Study of oncogenic transformation in ex vivo expanded mesenchymal cells, from paediatric bone marrow. Cell Prolif 2008;41(6):909-22.
26. Tarte K, Gaillard J, Lataillade JJ, Fouillard L, Becker M, Mossafa H, et al. Clinical-grade production of human mesenchymal stromal cells: occurrence of aneuploidy without transformation. Blood 2010;115(8):1549-53.
27. Gou S, Wang C, Liu T, Wu H, Xiong J, Zhou F, et al. Spontaneous differentiation of murine bone marrow-derived mesenchymal stem cells into adipocytes without malignant transformation after long-term culture. Cells Tissues Organs 2010;191(3):185-92.
28. de la Fuente R, Bernad A, Garcia-Castro J, Martin MC, Cigudosa JC. Retraction:
Spontaneous human adult stem cell transformation. Cancer Res 2010;70(16):6682.
29. Torsvik A, Rosland GV, Svendsen A, Molven A, Immervoll H, McCormack E, et al.
Spontaneous malignant transformation of human mesenchymal stem cells reflects cross- contamination: putting the research field on track - letter. Cancer Res 2010;70(15):6393- 30. Berger M, Muraro M, Fagioli F, Ferrari S. Osteosarcoma derived from donor stem cells 6.
carrying the Norrie’s disease gene. N Engl J Med 2008;359(23):2502-4.
31. Bielack SS, Rerin JS, Dickerhoff R, Dilloo D, Kremens B, von Stackelberg A, et al.
Osteosarcoma after allogeneic bone marrow transplantation. A report of four cases from the Cooperative Osteosarcoma Study Group (COSS). Bone Marrow Transplant 2003;31(5):353-9.
32. Berman SD, Calo E, Landman AS, Danielian PS, Miller ES, West JC, et al. Metastatic osteosarcoma induced by inactivation of Rb and p53 in the osteoblast lineage. Proc Natl Acad Sci U S A 2008;105(33):11851-6.
33. Lin PP, Pandey MK, Jin F, Raymond AK, Akiyama H, Lozano G. Targeted mutation of p53 and Rb in mesenchymal cells of the limb bud produces sarcomas in mice.
Carcinogenesis 2009;30(10):1789-95.
34. Mutsaers AJ, Ng AJ, Baker EK, Russell MR, Chalk AM, Wall M, et al. Modeling distinct osteosarcoma subtypes in vivo using Cre:lox and lineage-restricted transgenic shRNA.
Bone 2013;55(1):166-78.
27
1
General introduction and outline of this thesis
35. Walkley CR, Qudsi R, Sankaran VG, Perry JA, Gostissa M, Roth SI, et al. Conditional mouse osteosarcoma, dependent on p53 loss and potentiated by loss of Rb, mimics the human disease. Genes Dev 2008;22(12):1662-76.
36. Quist T, Jin H, Zhu JF, Smith-Fry K, Capecchi MR, Jones KB. The impact of osteoblastic differentiation on osteosarcomagenesis in the mouse. Oncogene 2015;34(32):4278-84.
37. Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 2002;108(1):17-29.
38. Pollak M. The insulin and insulin-like growth factor receptor family in neoplasia: an update. Nat.Rev.Cancer 2012;12(3):159-169.
39. Baxter RC. IGF binding proteins in cancer: mechanistic and clinical insights. Nat Rev Cancer 2014;14(5):329-41.
40. Duan C, Xu Q. Roles of insulin-like growth factor (IGF) binding proteins in regulating IGF actions. Gen Comp Endocrinol 2005;142(1-2):44-52.
41. Knudsen L, De Meyts P, Kiselyov VV. Insight into the molecular basis for the kinetic differences between the two insulin receptor isoforms. Biochem J 2011;440(3):397-403.
42. Siddle K. Molecular basis of signaling specificity of insulin and IGF receptors: neglected corners and recent advances. Front Endocrinol.(Lausanne) 2012;3:34.
43. Siddle K. Signalling by insulin and IGF receptors: supporting acts and new players.
J.Mol.Endocrinol. 2011;47(1):R1-10.
44. Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell 2007;12(1):9-22.
45. Liu JP, Baker J, Perkins AS, Robertson EJ, Efstratiadis A. Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r).
Cell 1993;75(1):59-72.
46. Maki RG. Small is beautiful: insulin-like growth factors and their role in growth, development, and cancer. J Clin Oncol 2010;28(33):4985-95.
47. Arora RS, Kontopantelis E, Alston RD, Eden TO, Geraci M, Birch JM. Relationship between height at diagnosis and bone tumours in young people: a meta-analysis. Cancer Causes Control 2011;22(5):681-8.
48. Mirabello L, Pfeiffer R, Murphy G, Daw NC, Patino-Garcia A, Troisi RJ, et al. Height at diagnosis and birth-weight as risk factors for osteosarcoma. Cancer Causes Control 2011;22(6):899-908.
49. Lima GA, Gomes EM, Nunes RC, Vieira Neto L, Sieiro AP, Brabo EP, et al.
Osteosarcoma and acromegaly: a case report and review of the literature. J Endocrinol Invest 2006;29(11):1006-11.
50. Steuerman R, Shevah O, Laron Z. Congenital IGF1 deficiency tends to confer protection against post-natal development of malignancies. Eur J Endocrinol 2011;164(4):485-9.
51. Ergun-Longmire B, Mertens AC, Mitby P, Qin J, Heller G, Shi W, et al. Growth hormone treatment and risk of second neoplasms in the childhood cancer survivor. J Clin Endocrinol Metab 2006;91(9):3494-8.
52. Bakker B, Oostdijk W, Geskus RB, Stokvis-Brantsma WH, Vossen JM, Wit JM. Growth hormone (GH) secretion and response to GH therapy after total body irradiation and haematopoietic stem cell transplantation during childhood. Clin Endocrinol (Oxf) 2007;67(4):589-97.
53. Behjati S, Tarpey PS, Haase K, Ye H, Young MD, Alexandrov LB, et al. Recurrent mutation of IGF signalling genes and distinct patterns of genomic rearrangement in osteosarcoma. Nat Commun 2017;8:15936.
28 Chapter 1
54. van Maldegem AM, Bovee JV, Peterse EF, Hogendoorn PC, Gelderblom H. Ewing sarcoma: The clinical relevance of the insulin-like growth factor 1 and the poly-ADP- ribose-polymerase pathway. Eur J Cancer 2016;53:171-80.
55. Mirabello L, Troisi RJ, Savage SA. Osteosarcoma incidence and survival rates from 1973 to 2004: data from the Surveillance, Epidemiology, and End Results Program.
Cancer 2009;115(7):1531-43.
56. van Oosterwijk JG, Anninga JK, Gelderblom H, Cleton-Jansen AM, Bovee JV. Update on targets and novel treatment options for high-grade osteosarcoma and chondrosarcoma.
Hematol Oncol Clin North Am 2013;27(5):1021-48.
57. Rosenberg AE, Cleton-Jansen A-M, de Pinieux G, Deyrup AT, Hauben E, Squire J.
Conventional osteosarcoma. In: Fletcher CDM, Bridge JA, Hogendoorn PCW, Mertens F, editors. WHO Classification of Tumours of Soft Tissue and Bone: IARC: Lyon; 2013.
p. 282-288.
58. Evans HL, Ayala AG, Romsdahl MM. Prognostic factors in chondrosarcoma of bone: a clinicopathologic analysis with emphasis on histologic grading. Cancer 1977;40(2):818- 59. Gelderblom H, Hogendoorn PC, Dijkstra SD, van Rijswijk CS, Krol AD, Taminiau AH, 31.
et al. The clinical approach towards chondrosarcoma. Oncologist. 2008;13(3):320-329.
60. Hogendoorn PCW, Bovée JVMG, Nielsen GP. Chondrosarcoma (grades I-III), including primary and secondary variants and periosteal chondrosarcoma. In: Fletcher CDM, Bridge JA, Hogendoorn PCW, Mertens F, editors. WHO Classification of Tumours of Soft Tissue and Bone: IARC: Lyon; 2013. p. 264-268.
61. Duchman KR, Lynch CF, Buckwalter JA, Miller BJ. Estimated cause-specific survival continues to improve over time in patients with chondrosarcoma. Clin Orthop Relat Res 2014;472(8):2516-25.
62. van Maldegem AM, Gelderblom H, Palmerini E, Dijkstra SD, Gambarotti M, Ruggieri P, et al. Outcome of advanced, unresectable conventional central chondrosarcoma. Cancer 2014.
63. Italiano A, Mir O, Cioffi A, Palmerini E, Piperno-Neumann S, Perrin C, et al. Advanced chondrosarcomas: role of chemotherapy and survival. Ann Oncol 2013;24(11):2916-22.
64. Flanagan AM, Lindsay D. A diagnostic approach to bone tumours. Pathology 2017.
65. Amary MF, Damato S, Halai D, Eskandarpour M, Berisha F, Bonar F, et al. Ollier disease and Maffucci syndrome are caused by somatic mosaic mutations of IDH1 and IDH2. Nat Genet 2011;43(12):1262-5.
66. Pansuriya TC, van Eijk R, d’Adamo P, van Ruler MA, Kuijjer ML, Oosting J, et al. Somatic mosaic IDH1 and IDH2 mutations are associated with enchondroma and spindle cell hemangioma in Ollier disease and Maffucci syndrome. Nat Genet 2011;43(12):1256-61.
67. Amary MF, Bacsi K, Maggiani F, Damato S, Halai D, Berisha F, et al. IDH1 and IDH2 mutations are frequent events in central chondrosarcoma and central and periosteal chondromas but not in other mesenchymal tumours. J Pathol 2011;224(3):334-43.
68. Xu W, Yang H, Liu Y, Yang Y, Wang P, Kim SH, et al. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of alpha-ketoglutarate-dependent dioxygenases. Cancer Cell 2011;19(1):17-30.
69. Noushmehr H, Weisenberger DJ, Diefes K, Phillips HS, Pujara K, Berman BP, et al.
Identification of a CpG island methylator phenotype that defines a distinct subgroup of glioma. Cancer Cell 2010;17(5):510-22.
29
1
General introduction and outline of this thesis
70. Christensen BC, Smith AA, Zheng S, Koestler DC, Houseman EA, Marsit CJ, et al.
DNA methylation, isocitrate dehydrogenase mutation, and survival in glioma. J Natl Cancer Inst 2011;103(2):143-53.
71. Laffaire J, Everhard S, Idbaih A, Criniere E, Marie Y, de Reynies A, et al. Methylation profiling identifies 2 groups of gliomas according to their tumorigenesis. Neuro Oncol 2011;13(1):84-98.
72. Turcan S, Rohle D, Goenka A, Walsh LA, Fang F, Yilmaz E, et al. IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature 2012;483(7390):479- 73. Figueroa ME, Abdel-Wahab O, Lu C, Ward PS, Patel J, Shih A, et al. Leukemic IDH1 83.
and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell 2010;18(6):553-67.
74. Bovee JVMG, Heymann D, Wuyts W. Osteochondroma. World Health Organisation Classification of Tumours of Soft Tissue and Bone: IARC Press; Lyon, 2013, pp 273- 75. Jennes I, Pedrini E, Zuntini M, Mordenti M, Balkassmi S, Asteggiano CG, et al. Multiple 274.
osteochondromas: mutation update and description of the multiple osteochondromas mutation database (MOdb). Hum Mutat 2009;30(12):1620-7.
76. Wuyts W, Van Hul W, De Boulle K, Hendrickx J, Bakker E, Vanhoenacker F, et al.
Mutations in the EXT1 and EXT2 genes in hereditary multiple exostoses. Am J Hum Genet 1998;62(2):346-54.
77. de Andrea CE, Wiweger M, Prins F, Bovee JV, Romeo S, Hogendoorn PC. Primary cilia organization reflects polarity in the growth plate and implies loss of polarity and mosaicism in osteochondroma. Lab Invest 2010;90(7):1091-101.
78. Szuhai K, Jennes I, de Jong D, Bovee JV, Wiweger M, Wuyts W, et al. Tiling resolution array-CGH shows that somatic mosaic deletion of the EXT gene is causative in EXT gene mutation negative multiple osteochondromas patients. Hum Mutat 2011;32(2):E2036- 79. de Andrea CE, Reijnders CM, Kroon HM, de Jong D, Hogendoorn PC, Szuhai K, et 49.
al. Secondary peripheral chondrosarcoma evolving from osteochondroma as a result of outgrowth of cells with functional EXT. Oncogene 2012;31(9):1095-104.
80. Stickens D, Brown D, Evans GA. EXT genes are differentially expressed in bone and cartilage during mouse embryogenesis. Dev Dyn 2000;218(3):452-64.
81. Jochmann K, Bachvarova V, Vortkamp A. Heparan sulfate as a regulator of endochondral ossification and osteochondroma development. Matrix Biol 2014;34:55-63.
82. Chung UI, Lanske B, Lee K, Li E, Kronenberg H. The parathyroid hormone/parathyroid hormone-related peptide receptor coordinates endochondral bone development by directly controlling chondrocyte differentiation. Proc Natl Acad Sci U S A 1998;95(22):13030-5.
83. Chung UI, Schipani E, McMahon AP, Kronenberg HM. Indian hedgehog couples chondrogenesis to osteogenesis in endochondral bone development. J Clin Invest 2001;107(3):295-304.
84. Inwards C, Hogendoorn PCW. Dedifferentiated chondrosarcoma. In: Fletcher CDM, Bridge JA, Hogendoorn PCW, Mertens F, editors. WHO Classification of Tumours of Soft Tissue and Bone: IARC: Lyon; 2013. p. 269-270.
85. Grimer RJ, Gosheger G, Taminiau A, Biau D, Matejovsky Z, Kollender Y, et al.
Dedifferentiated chondrosarcoma: prognostic factors and outcome from a European group. Eur J Cancer 2007;43(14):2060-5.
30 Chapter 1
86. Meijer D, de Jong D, Pansuriya TC, van den Akker BE, Picci P, Szuhai K, et al. Genetic characterization of mesenchymal, clear cell, and dedifferentiated chondrosarcoma.
Genes Chromosomes Cancer 2012;51(10):899-909.
87. Morrison C, Radmacher M, Mohammed N, Suster D, Auer H, Jones S, et al. MYC amplification and polysomy 8 in chondrosarcoma: array comparative genomic hybridization, fluorescent in situ hybridization, and association with outcome. J Clin Oncol 2005;23(36):9369-76.
88. Gao L, Hong X, Guo X, Cao D, Gao X, DeLaney TF, et al. Targeted next-generation sequencing of dedifferentiated chondrosarcoma in the skull base reveals combined TP53 and PTEN mutations with increased proliferation index, an implication for pathogenesis.
Oncotarget 2016;7(28):43557-43569.
89. Nakashima Y, de Pinieux G, Ladanyi M. Mesenchymal chondrosarcoma. In: Fletcher CDM, Bridge JA, Hogendoorn PCW, Mertens F, editors. WHO Classification of Tumours of Soft Tissue and Bone: IARC: Lyon; 2013. p. 271-272.
90. Frezza AM, Cesari M, Baumhoer D, Biau D, Bielack S, Campanacci DA, et al.
Mesenchymal chondrosarcoma: prognostic factors and outcome in 113 patients. A European Musculoskeletal Oncology Society study. Eur J Cancer 2015;51(3):374-81.
91. Xu J, Li D, Xie L, Tang S, Guo W. Mesenchymal chondrosarcoma of bone and soft tissue:
a systematic review of 107 patients in the past 20 years. PLoS One 2015;10(4):e0122216.
92. Schneiderman BA, Kliethermes SA, Nystrom LM. Survival in Mesenchymal Chondrosarcoma Varies Based on Age and Tumor Location: A Survival Analysis of the SEER Database. Clin Orthop Relat Res 2017;475(3):799-805.
93. Cesari M, Bertoni F, Bacchini P, Mercuri M, Palmerini E, Ferrari S. Mesenchymal chondrosarcoma. An analysis of patients treated at a single institution. Tumori 2007;93(5):423-7.
94. Dantonello TM, Int-Veen C, Leuschner I, Schuck A, Furtwaengler R, Claviez A, et al.
Mesenchymal chondrosarcoma of soft tissues and bone in children, adolescents, and young adults: experiences of the CWS and COSS study groups. Cancer 2008;112(11):2424-31.
95. Bae DK, Park YK, Chi SG, Lee CW, Unni KK. Mutational alterations of the p16CDKN2A tumor suppressor gene have low incidence in mesenchymal chondrosarcoma. Oncol Res 2000;12(1):5-10.
96. Park YK, Park HR, Chi SG, Kim CJ, Sohn KR, Koh JS, et al. Overexpression of p53 and rare genetic mutation in mesenchymal chondrosarcoma. Oncol Rep 2000;7(5):1041-7.
97. Wang L, Motoi T, Khanin R, Olshen A, Mertens F, Bridge J, et al. Identification of a novel, recurrent HEY1-NCOA2 fusion in mesenchymal chondrosarcoma based on a genome-wide screen of exon-level expression data. Genes Chromosomes Cancer 2012;51(2):127-39.
98. Nyquist KB, Panagopoulos I, Thorsen J, Haugom L, Gorunova L, Bjerkehagen B, et al.
Whole-transcriptome sequencing identifies novel IRF2BP2-CDX1 fusion gene brought about by translocation t(1;5)(q42;q32) in mesenchymal chondrosarcoma. PLoS One 2012;7(11):e49705.
99. McCarthy EF, Hogendoorn PC. Clear Cell Chondrosarcoma. World Health Organisation Classification of Tumours of Soft Tissue and Bone: IARC Press; Lyon, 2013, pp 273- 100. Bjornsson J, Unni KK, Dahlin DC, Beabout JW, Sim FH. Clear cell chondrosarcoma of 274.
bone. Observations in 47 cases. Am J Surg Pathol 1984;8(3):223-30.
101. Present D, Bacchini P, Pignatti G, Picci P, Bertoni F, Campanacci M. Clear cell chondrosarcoma of bone. A report of 8 cases. Skeletal Radiol 1991;20(3):187-91.
31
1
General introduction and outline of this thesis
102. Boehme KA, Schleicher SB, Traub F, Rolauffs B. Chondrosarcoma: A Rare Misfortune in Aging Human Cartilage? The Role of Stem and Progenitor Cells in Proliferation, Malignant Degeneration and Therapeutic Resistance. Int J Mol Sci 2018;19(1).
103. Aigner T. Towards a new understanding and classification of chondrogenic neoplasias of the skeleton--biochemistry and cell biology of chondrosarcoma and its variants.
Virchows Arch 2002;441(3):219-30.
104. Suijker J, Baelde HJ, Roelofs H, Cleton-Jansen AM, Bovee JV. The oncometabolite D-2- hydroxyglutarate induced by mutant IDH1 or -2 blocks osteoblast differentiation in vitro and in vivo. Oncotarget 2015;6(17):14832-42.
105. Wiweger MI, de Andrea CE, Scheepstra KW, Zhao Z, Hogendoorn PC. Possible effects of EXT2 on mesenchymal differentiation--lessons from the zebrafish. Orphanet J Rare Dis 2014;9:35.
106. Jones KB, Piombo V, Searby C, Kurriger G, Yang B, Grabellus F, et al. A mouse model of osteochondromagenesis from clonal inactivation of Ext1 in chondrocytes. Proc Natl Acad Sci U S A 2010;107(5):2054-9.
107. Matsumoto K, Irie F, Mackem S, Yamaguchi Y. A mouse model of chondrocyte-specific somatic mutation reveals a role for Ext1 loss of heterozygosity in multiple hereditary exostoses. Proc Natl Acad Sci U S A 2010;107(24):10932-7.
108. Bovee JV, Cleton-Jansen AM, Taminiau AH, Hogendoorn PC. Emerging pathways in the development of chondrosarcoma of bone and implications for targeted treatment. Lancet Oncol 2005;6(8):599-607.
109. Boeuf S, Kunz P, Hennig T, Lehner B, Hogendoorn P, Bovee J, et al. A chondrogenic gene expression signature in mesenchymal stem cells is a classifier of conventional central chondrosarcoma. J Pathol 2008;216(2):158-66.
110. Warburg O. On the origin of cancer cells. Science 1956;123(3191):309-14.
111. Warburg O, Posener K, Negelein E. Üeber den Stoffwechsel der Tumoren. Biochem Z 1924;152:319-344.
112. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 2009;324(5930):1029-33.
113. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144(5):646-74.
114. Rohle D, Popovici-Muller J, Palaskas N, Turcan S, Grommes C, Campos C, et al. An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells.
Science 2013;340(6132):626-30.
115. Suijker J, Oosting J, Koornneef A, Struys EA, Salomons GS, Schaap FG, et al. Inhibition of mutant IDH1 decreases D-2-HG levels without affecting tumorigenic properties of chondrosarcoma cell lines. Oncotarget 2015;6(14):12505-19.
116. Wang F, Travins J, DeLaBarre B, Penard-Lacronique V, Schalm S, Hansen E, et al.
Targeted inhibition of mutant IDH2 in leukemia cells induces cellular differentiation.
Science 2013;340(6132):622-6.
117. Chen C, Liu Y, Lu C, Cross JR, Morris JPt, Shroff AS, et al. Cancer-associated IDH2 mutants drive an acute myeloid leukemia that is susceptible to Brd4 inhibition. Genes Dev 2013;27(18):1974-85.
118. Turcan S, Fabius AW, Borodovsky A, Pedraza A, Brennan C, Huse J, et al. Efficient induction of differentiation and growth inhibition in IDH1 mutant glioma cells by the DNMT Inhibitor Decitabine. Oncotarget 2013;4(10):1729-36.
32 Chapter 1
119. Tateishi K, Wakimoto H, Iafrate AJ, Tanaka S, Loebel F, Lelic N, et al. Extreme Vulnerability of IDH1 Mutant Cancers to NAD+ Depletion. Cancer Cell 2015;28(6):773-84.
120. Hartmann C, Meyer J, Balss J, Capper D, Mueller W, Christians A, et al. Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse gliomas. Acta Neuropathol 2009;118(4):469-74.
121. Sanson M, Marie Y, Paris S, Idbaih A, Laffaire J, Ducray F, et al. Isocitrate dehydrogenase 1 codon 132 mutation is an important prognostic biomarker in gliomas. J Clin Oncol 2009;27(25):4150-4.
122. Nobusawa S, Watanabe T, Kleihues P, Ohgaki H. IDH1 mutations as molecular signature and predictive factor of secondary glioblastomas. Clin Cancer Res 2009;15(19):6002-7.
123. Cleven AHG, Suijker J, Agrogiannis G, Briaire-de Bruijn IH, Frizzell N, Hoekstra AS, et al. IDH1 or -2 mutations do not predict outcome and do not cause loss of 5-hydroxymethylcytosine or altered histone modifications in central chondrosarcomas.
Clin Sarcoma Res 2017;7:8.
124. Bovee JV, Hogendoorn PC, Wunder JS, Alman BA. Cartilage tumours and bone development: molecular pathology and possible therapeutic targets. Nat Rev Cancer 2010;10(7):481-8.
125. Ho L, Stojanovski A, Whetstone H, Wei QX, Mau E, Wunder JS, et al. Gli2 and p53 cooperate to regulate IGFBP-3- mediated chondrocyte apoptosis in the progression from benign to malignant cartilage tumors. Cancer Cell 2009;16(2):126-36.
126. Wu CM, Li TM, Hsu SF, Su YC, Kao ST, Fong YC, et al. IGF-I enhances alpha5beta1 integrin expression and cell motility in human chondrosarcoma cells. J Cell Physiol 2011;226(12):3270-7.
127. Zhang YX, van Oosterwijk JG, Sicinska E, Moss S, Remillard SP, van Wezel T, et al.
Functional profiling of receptor tyrosine kinases and downstream signaling in human chondrosarcomas identifies pathways for rational targeted therapy. Clin Cancer Res 2013;19(14):3796-807.
128. Tateishi K, Wakimoto H, Iafrate AJ, Tanaka S, Loebel F, Lelic N, et al. Extreme Vulnerability of IDH1 Mutant Cancers to NAD+ Depletion. Cancer Cell 2015;28(6):773-784.
129. Sampath D, Zabka TS, Misner DL, O’Brien T, Dragovich PS. Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) as a therapeutic strategy in cancer. Pharmacol Ther 2015;151:16-31.
130. Xiao Y, Elkins K, Durieux JK, Lee L, Oeh J, Yang LX, et al. Dependence of tumor cell lines and patient-derived tumors on the NAD salvage pathway renders them sensitive to NAMPT inhibition with GNE-618. Neoplasia 2013;15(10):1151-60.
131. Heyes MP, Chen CY, Major EO, Saito K. Different kynurenine pathway enzymes limit quinolinic acid formation by various human cell types. Biochem J 1997;326 ( Pt 2):351-6.
132. Grassian AR, Parker SJ, Davidson SM, Divakaruni AS, Green CR, Zhang X, et al.
IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism. Cancer Res 2014;74(12):3317-31.
133. Cuyas E, Fernandez-Arroyo S, Corominas-Faja B, Rodriguez-Gallego E, Bosch-Barrera J, Martin-Castillo B, et al. Oncometabolic mutation IDH1 R132H confers a metformin- hypersensitive phenotype. Oncotarget 2015;6(14):12279-96.
134. van Lith SA, Navis AC, Verrijp K, Niclou SP, Bjerkvig R, Wesseling P, et al. Glutamate as chemotactic fuel for diffuse glioma cells: are they glutamate suckers? Biochim Biophys Acta 2014;1846(1):66-74.
33
1
General introduction and outline of this thesis
135. Seltzer MJ, Bennett BD, Joshi AD, Gao P, Thomas AG, Ferraris DV, et al. Inhibition of glutaminase preferentially slows growth of glioma cells with mutant IDH1. Cancer Res 2010;70(22):8981-7.
136. Metallo CM, Gameiro PA, Bell EL, Mattaini KR, Yang J, Hiller K, et al. Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia. Nature 2011;481(7381):380-4.
137. Reitman ZJ, Duncan CG, Poteet E, Winters A, Yan LJ, Gooden DM, et al. Cancer- associated isocitrate dehydrogenase 1 (IDH1) R132H mutation and d-2-hydroxyglutarate stimulate glutamine metabolism under hypoxia. J Biol Chem 2014;289(34):23318-28.
138. Matre P, Velez J, Jacamo R, Qi Y, Su X, Cai T, et al. Inhibiting glutaminase in acute myeloid leukemia: metabolic dependency of selected AML subtypes. Oncotarget 2016;7(48):79722-79735.
Chapter 2
In vitro studies of osteosarcoma:
A researcher’s perspective of quantity and quality
Elisabeth F.P. Peterse, Thed N. van Leeuwen, Anne-Marie Cleton-Jansen Journal of Bone Oncology, 2017, 7, 29-31
